IDE antibody (AA 491-590)

Catalog No. ABIN723680

The Rabbit Polyclonal anti-IDE antibody has been validated for WB, ELISA, IHC (p), IF (cc), IF (p) and IHC (fro) and has been independently validated. It is suitable to detect IDE in samples from Human, Mouse and Rat.

Quick Overview for IDE antibody (AA 491-590) (ABIN723680)

Target: IDE (Insulin-Degrading Enzyme (IDE))

Reactivity: Human, Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This IDE antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p)), Immunohistochemistry (Frozen Sections) (IHC (fro))

Product Details anti-IDE Antibody

Binding Specificity: AA 491-590

Cross-Reactivity: Human, Mouse, Rat

Predicted Reactivity: Cow,Pig,Chicken

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic peptide derived from human IDE

Isotype: IgG

Application Details

Application Notes: WB 1:300-5000
ELISA 1:500-1000
IHC-P 1:200-400
IHC-F 1:100-500
IF(IHC-P) 1:50-200
IF(IHC-F) 1:50-200
IF(ICC) 1:50-200

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #104435 (Immunohistochemistry)

by: Prof. Merighi, Laboratory of Neurobiology, Department of Veterinary Sciences, University of Turin

No.: #104435

Date: 03/15/2023

Antigen: IDE

Lot Number: 9C07M588

Method validated: Immunohistochemistry

Positive Control:

Adult mouse liver fixed in 4% paraformaldehyde

Negative Control:

One control slice for each experimental procedure processed omitting the primary antibody; overnight incubation in diluent solution only.

Notes:

Passed. The IDE antibody (AA 491-590) ABIN723680 works in IHC-P at 1:100 concentrations with Tyramide amplification.

Validation Images

Staining of IDE positive cells in the adult mouse liver with ABIN723680.

Full Methods

Primary Antibody: ABIN723680

Secondary Antibody: poly-HRP conjugated goat anti-rabbit antibody

Full Protocol:

• Perfuse mice with paraformaldehyde 4% in 0.1 M phosphate buffer pH 7.4 and post-fix in the same fixative for an additional 2 h at RT.
• Wash, dehydrate, and embed samples in paraffin wax.
• Wash several times with 0.01 M PBS.
• Cut liver with a microtome into 20 µm sections and mount on glass slides.
• After paraffin removal, incubate sections for 1 h at RT in PBS containing 1% albumin from chicken egg white (Sigma, A5378) and 0.3% Triton-X-100 (BioRad, 161-0407, lot 00583) to block non-specific binding sites.
• Incubate sections with primary rabbit anti-IDE (antibodies-online, ABIN723680, lot 9C07M588) diluted 1:50, 1:100, 1:200, and 1:300 in PBS-BSA-PLL ON at RT in a humid chamber.
• Wash sections 3x 5 min with 0.01 M PBS.
• Incubate sections with secondary poly-HRP conjugated goat anti-rabbit antibody from Alexa Fluor 488 Tyramide SuperBoost Kit, goat anti-rabbit IgG (Thermo Fisher Scientific, B40922, lot 2465062) for 1 h at RT.
• Wash sections 3x 5 min with 0.01 M PBS.
• Incubate sections with Tyramide working solution containing 100X Tyramide stock solution (Alexa 488), 100X H₂O₂ solution and 1X Reaction buffer for 10 min.
• Stop the reaction with the Reaction Stop Reagent working solution.
• Wash sections 3x 5 min with 0.01M PBS.
• Mount specimens in Fluoroshield (Sigma, F6182, lot MKCB0153V).
• Acquire images with a fluorescence microscope and appropriate filter settings for AF488, e.g. Leica DM 6000B fluorescence microscope equipped with a digital camera at 40x magnification.

Independent Validation (WB)

Validation #104497 (Western Blotting)

by: Prof. Merighi, Laboratory of Neurobiology, Department of Veterinary Sciences, University of Turin

No.: #104497

Date: 03/15/2023

Antigen: IDE

Lot Number: 9C07M588

Method validated: Western Blotting

Positive Control:

Adult mouse brain, cerebellum, and liver

Notes:

Passed. The IDE antibody (AA 491-590) ABIN723680 works in WB at 1:1000 concentrations with sensitive ECL substrate.

Validation Images

Western blot detection of IDE (110 kDa) in adult mouse brain (1), cerebellum (2), and liver (3) tissue homogenates with ABIN723680.

Full Methods

Primary Antibody: ABIN723680

Secondary Antibody: HRP-conjugated mouse anti-rabbit

Full Protocol:

• Homogenize tissues with cold lysis buffer containing 50 mM Tris HCl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 1% protease inhibitor (Sigma P8340) using an ultrasonic homogenizer (MSE, SoniPrep 150) with 16 amplitude, 20 s on, 10 s off pulse for 60 s.
• Centrifuge tissue homogentates at 13,000 rpm for 20 min at 4 °C.
• Collect supernatants and Determine total protein content using a Bradford assay.
• Denature 50 µg of total protein for 5 min at 90 °C and subsequently separate them on a denaturing 12% PAGE-SDS gel alongside a Precision Plus Protein Dual Color Standard (Bio-Rad, 160374).
• Electro-transfer proteins onto nitrocellulose membrane (Amerscham Biosciences, RPN203D) ON in the cold room.
• Wash membrane 3x for 10 mon with 0.01 M PBS containing 0.1% Tween-20 (PBST).
• Block membrane with PBST containing 2% bovine serum albumin for 1 h at RT.
• Incubate membrane with primary rabbit anti-IDE antibody (antibodies-online, ABIN723680, lot 9C07M588) diluted 1:1,000 in PBST ON at 4 °C.
• Wash membrane 3x 10 min with PBST.
• Incubate membrane with secondary HRP-conjugated mouse anti-rabbit IgG (Sigma, A1949) diluted 1:4,000 in PBST for 1 h at RT.
• Wash membrane 3x 10 min with PBST.
• Visualize proteins with WesternBright Sirius HRP substrate (Advansta, K-12043) using a ChemiDoc Imaging System.

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: 0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.

Expiry Date: 12 months

Target Details for IDE

Target: IDE (Insulin-Degrading Enzyme (IDE))

Alternative Name: IDE

Background:

Synonyms: INSULYSIN, Insulin-degrading enzyme, Abeta-degrading protease, Insulin protease, Insulinase, IDE

Background: Plays a role in the cellular breakdown of insulin, IAPP, glucagon, bradykinin, kallidin and other peptides, and thereby plays a role in intercellular peptide signaling. Degrades amyloid formed by APP and IAPP. May play a role in the degradation and clearance of naturally secreted amyloid beta-protein by neurons and microglia.

Gene ID: 3416

UniProt: P14735

Pathways: SARS-CoV-2 Protein Interactome